Enamel is the hardest substance in the human body. Fluoride is commonly used in caries prevention, but high levels of fluoride result in fluorosis and enamel defects. Over several decades researchers have defined the biochemical and structural biology behind fluorotic enamel, however, a gap exists in relating these changes to the molecular signaling events that direct amelogenesis. The goal of these studies is to identify signaling pathways that are altered upon exposure to fluoride thereby resulting in amelognesis defects using a novel fluorosis model: the zebrafish. The zebrafish offers a unique opportunity to answer some of the molecular signaling questions relevant to the mechanism of human fluorosis because they mature rapidly, replace their teeth continuously and possess many of the same signaling pathways as described for human tooth development. Zebrafish teeth display similar fluorotic defects to those seen in mammals and compositional analysis reveals that zebrafish fluorosis is concomitant with decreased mineral content, and increased protein levels similar to that seen in humans. Furthermore, the elevated level of apoptosis in the tooth buds of the fluoride treated animals, correspond with altered cell signaling events indicated by decreased TGF-beta signaling pathway components. Specific Aim 1 will utilize microarray technology to identify genes whose expression is altered in fluorotic teeth. Specific Aim 2 will localize the changes in gene expression in subpopulations of tooth bud cells using laser microdissection and Real Time- PCR. Specific Aim 3 will further characterize the structural changes in fluorotic zebrafish enameloid. Our hypothesis is that fluoride alters growth factor receptor signaling, which perturbs ameloblast activities, resulting in increased programmed cell death and defective mineralization of the enameloid-matrix. The studies performed in this work will provide a foundation for our long-term goals, which include analysis of molecular signaling events regulating enamel formation in mammals.